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Effects of electronic cigarettes on viability and proliferation of lung mesenchymal stem cells

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Effects of electronic cigarettes on viability and proliferation of lung mesenchymal stem cells

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Content Effects of electronic cigarettes on viability and proliferation
of lung mesenchymal stem cells
By
Krysta Marie Siu
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR MEDICINE)
December 2023
Copyright 2023 Krysta Marie Siu

ii
Acknowledgements
I would like to thank my mentor Dr. Ching-Ling (Ellen) Lien for her help and guidance and
allowing me to do my research as a part of her lab. I would also like to thank my thesis committee
Dr. Judd Rice and Dr. Mark Frey for all their help and advice to help me finish this thesis. I would
also like to thank everyone in the lab: Yuhan Sun, Xidi Feng, Subir Kapuria, Stanislao Travisano,
and Raquel Rodriguez for their support and a special thanks to Siqi Tao for all her help and
guidance in my research. Finally, I would like to thank my father, Gilberto Siu, and my significant
other, Cesar Patino, for all their support throughout all my time in school. If I did not have a support
system like the two of them, I would not have been able to push through and finish my research or
Masters.

iii
Table of Contents
ACKNOWLEDGEMENTS ………………………………………………….………….……...…ii
LIST OF FIGURES ……………………………………..………………….……………..………iv
ABSTRACT ………………………………………………………….…………………..….…….v
Chapter One: INTRODUCTION ……………………………………………………..……...…….1
Chronic Obstructive Pulmonary Disease (COPD) …………………………..…….……….1
Electronic cigarettes ……………………………………………………………….….……2
Lung mesenchymal stem cells …………………………………….…………………….…3
TBX4 …………………………………………………………….……….…..……………4
Triple Transgenic Mice ……………………………………..……….………..……………4
GFP and RFP Lung mesenchymal stem cells ……………..……………….…….…………5
Previous studies …………………………………………………..……………..…………5
Chapter Two: MATERIALS AND METHODS ……………………………………….………...…6
Cell culture ………………………………………………………………………..….……6
Viability ………………...…………………………………………………….……………6
Proliferation ……………………………………………………………………....…..……7
Calculations ……………………………………………………………………………..…7
Chapter Three: RESULTS …………………………………………………………………………8
Treatment of nicotine, PGVG, and E-Cig ……..………………………….....……....….....8
Viability ……………………………………………………………………..……………12
Proliferation ………………………………………………………………....……………15
Chapter Four: DISCUSSION………………………………………………….…………………..17
Viability ………………………………………………………………………..…………17
Proliferation ………………………………………………………………..………..……17
General…………………………………………………………………………...….……17
Chapter Five: FUTURE STUDY ………………………………………………………....………19
REFERENCES ………………………………………………………………….…………….…20

iv
List of figures
Figure 1: Dose dependance data Lower concentrations…………………….…………………….9
Figure 2: Dose dependence data Expanded Range and hours………………..………..…………10
Figure 3: Dose dependance data narrowing concentrations.……………….……..….…..……….11
Figure 4: MTT Viability after 48 hours ………………………………………….………………13
Figure 5: Effects of different flavorings.………………………………………….…..…..………14
Figure 6: Cinnamaldehyde broken plate…………………………………….………..…….……14
Figure 7: EdU assay to determine cell proliferation.…………………………………..…...……16

v
Abstract
For years now electronic cigarettes have been promoted as a better alternative to smoking
cigarettes, but the effects of electronic cigarettes have not been fully explored. Smoking can lead
to diseases such as Chronic obstructive pulmonary disease (COPD). When tissues in the body are
damaged, such as the lungs, mesenchymal stem cells (MSC) help repair and regenerate cells.
Understanding the effects of electronic cigarettes on the lung mesenchymal stem cells will help
us understand the relationship between smoking electronic cigarettes and diseases such as
emphysema and chronic bronchitis. The MSCs help keep the lungs in homeostasis and aid in
organogenesis. If the viability is decreasing due to the exposure to E-Cigs, then the MSCs will
not be able to continue to aid in homeostasis. Using Lung mesenchymal stem cells from triple
transgenic mice we were able to test the viability and proliferation of Tbx4 lineage labeled fetal
lung-derived MSCs and Tbx4 negative MSCs.

1
Chapter One: Introduction
Chronic Obstructive Pulmonary Disease (COPD)
The use of tobacco and nicotine products is the leading cause of preventable deaths around the
world. Smoking such products can lead to several complications and diseases such as Ecigarette or vaping product use-associated lung injury (EVALI) and Chronic obstructive
pulmonary disease (COPD) [8,14]. COPD is a group of lung diseases, such as emphysema and
chronic bronchitis, that block airflow by damaging the lungs or the overproducing phlegm
making it difficult to breathe. COPD affects over 15 million adults in the unites states alone.
Exposure to air pollutants or other irritants can lead to COPD as well as having an Alpha-1
antitrypsin (AAT) deficiency. AAT deficiency is a genetic condition where the body does not
produce enough ATT. ATT is produced in the liver, it helps protect the lungs and having a
deficiency can heighten your risk of having COPD and having cirrhosis. Although there are
ways of getting COPD without smoking over 70% of patients living with COPD are chronic
smokers. Some symptoms of COPD include having a chronic cough, overproduction of mucus,
fatigue, and dyspnea.

2
Electronic cigarettes
Since the invention and release of electronic cigarettes (E-cigs) in 2003, they have been sold as a
less harmful alternative to conventional cigarettes [13]. They have been seen as a better
alternative possibly due to the reduction in chemical compounds that are being introduced to the
body by smoking when compared to conventional cigarettes. Cigarette smoke can include over
7000 chemical compounds ranging from nitrogen, carbon monoxide, cyanide, and tar to name a
few [15]. E-cigs chemically are made up of primarily propylene glycol and vegetable glycerin
(PGVG), with differing concentrations of nicotine ranging from 0-50mg/mL and an optional
flavoring [8]. The nicotine content tends to vary between 3-36 mg/mL in more recent years the
nicotine content has gone up to 60 mg/ml using nicotine salts [7]. The E-cig “juice” are
vaporized using a power source and a heating element (atomizer). The atomizer is made up of a
coil (heats and vaporizes the E-cig “juice”) and a wick (typically made of cotton, used to draw
the “juice” to the coil). [11]. One of the biggest selling points for E-Cigs is they have been said
to help people who are highly addicted to nicotine quit their addiction, but that does not account
for the millions of people who are becoming addicted to smoking E-Cigs every year. Where now
it is approximated t that 4.5 % of adults are a current E-Cig user. Yet there is a lot that is still
unknown about these products and how they affect the human body. E-Cigs are also known to be
highly addictive especially with the higher concentrations of nicotine, the different flavors, and
accessibility of them. When it comes to conventional cigarettes typically menthol is one of the
only flavorings whereas E-cigarettes you have a myriad of flavors to choose from although the
effects are still not fully understood. In places such as the United States E-cigs can be bought in
concentrations of 5% (50mg/mL) nicotine, however, in places like Europe and Canada E-Cigs
nicotine concentrations are regulated and capped at 20mg/mL (2%.)

3
Lung mesenchymal stem cells
Mesenchymal stem cells (MSC) are multipotent stem cells found throughout the body. MSCs are
known for their regenerative properties and their ability to repair damaged tissue. MSC are found
in many tissues throughout the body. have various unique properties such as self-renewal,
immunosuppressive potential, and the ability to trans-differentiate. One of the unique
regenerative abilities of MSC to damaged cells though mitochondrial transfer. In mice and
humans MSC express CD44, CD166, CD105, and CD90 and are negative for CD34 and CD45
[4]. MSC cells also have the potential to differentiate into Osteoblast, adipocytes, and
chondrocytes. [2,4]. It was seen when epithelial cells void of mitochondria were co-cultured with
MSC’s, mitochondrial respiration was restored in the epithelial cells [12]. Lung mesenchymal
stem cells play a pivotal role working with the lung epithelial cells in lung organogenesis and
homeostasis [17].

4
TBX4 lung enhancer
T-Box factor 4 (TBX4) is part of the T-box transcription factor family which is known to aid in
the development of embryos. Previously TBX4 had been associated primarily with limb pattern
development, yet in 2013 TBX4 variants have been associated with a wide variety of lung
disorders [5]. Specifically in intron 3 there is a 5.5kb DNA segment that is conserved not only
throughout mammalian species, but also in fish that have lung like structures such as the
Coelacanth. That same segment is not present in other fish that do not develop lung like
structures suggesting the roll of that segment has been important to the development of air
breathing organs throughout early evolution [9,17].
Triple Transgenic Mice
Zhang et al. were able to create a transgenic mouse line with a Tbx4 lung enhancer drivenreverse tetracycline trans activator (tbx4-rtTA). Once crossed the tet-O-Cre and with a loxpmTomato-STOP-loxp-mGFP (mT-mG) reporter mice to generate triple transgenic mice, they
were able to target lung mesenchymal stem cells at different developmental stages. To further
identify different types of cells during different developmental time points, they induced the
expression of the TetO-Cre with the addition of doxycycline (Dox) thus resulting in detectable
mGFP expression due to the floxed-mTomato deletion when Cre recombination is induced.
Therefore, when the cells are Cre-negative they express mTomato [17]. In short if the MSC’s
were to fluoresce green they were derived from the Tbx4 lineage labeled trachea and lung
mesenchyme(mG or GFP cells for short), whereas if they fluoresced red that meant they were not
derived from the Tbx4 labeled lineage (mT or RFP cells for short).

5
GFP and RFP Lung mesenchymal stem cells
The lung mesenchymal stem cells were separated into cells from the airways and the vasculature
during embryonic development (GFP-MSC), and those derived from other tissues (RFP_MSC).
The lung mesenchymal cells were extracted from 6month old mice, cultured, and separated with
FACs sorting. Both cells expressed Sca1, CD90, and CD105 which are mesenchymal stem cell
markers and were negative for CD31, CD45, and CD326 which are not expressed in MSC but
expressed in endothelial cells. As the mice develop the ratio of which mG+ and mT+ cells are
present go from majority mG+ cells to roughly a 50/50 split of mG+ and mT+ cells when the
mice are 6 months old.
Previous studies
Nicotine has been found to decrease viability in certain cell lines such as keratinocytes and
hepatocytes in concentrations as low as 0.1 µL /mL (. In other cell lines such as cardiomyocytes,
it does not start to have significant decrease in viability until 10u µL /mL [1]. In cell lines such as
MRC-5, a lung fibroblast cell line, viability of the cells started to significantly decrease after 750
µMol (0.0046 µL/mL) [16]. Morris et al, tested the effects of 30 flavoring chemicals on human
epithelial cells and found many of had effects such as reduction in viability, and increase in ROS
and overall varying levels of cytotoxicity [10]. Kerasioti suggested the flavoring is more
indicative of toxicity than nicotine [6]

6
Chapter Two: Materials and Methods
Cell culture:
Two cell lines were received from Dr. Wei Shi’s Lab (University of Cincinnati). The lung
mesenchymal stem cells were obtained from 6-month-old triple transgenic mice. FACS sorting
was used to separate GFP (fetal lung derived) MSC and RFP (derived from other organs) MSC.
Cells were received at passage 21 for GFP and Passage 28 for RFP. GFP and RFP were seeded
and cultured separately. The cells were kept at 37 °C and 5% CO2. For all experiments the cells
were grown to ~80% confluency in 100mm culture plates. 10-022-CV Corning media
supplemented with 20% FBS, 1000 u/ml penicillin, 2mM Glutamine,1/1000 2-Me, and 1/1000
Dex.
Viability
MTT assay was performed to test viability. The MTT assay uses the mitochondria to change
tetrazolium from a yellow color to a purple color showing cellular metabolic activity. Cells were
seeded at 20% confluency in 96 well plates. The cells were left overnight after seeding. Nicotine,
PGVG, and a mixture of PGVG and nicotine that mimic E-Cigs were used and combined with
prepared media were used to treat the cells at different concentrations and for different time
points. After the desired time (24, 48, and 96 hours) the treated media was removed. The cells
were then washed with PBS, this step is crucial when working with PGVG as the PGVG would
interact with the MTT dye. Fresh media (100 µL) was then added to the cells after the removal of
the PBS. MTT dye (15 µL) was then added to each well. Once the wells had dye, the plates were
left in the incubator at 37 degrees and 5% CO2 for 1-4 hours (2 hours was typically used).
Enough time need to be allotted to allow the cells to break down the dye. Once the incubation
period was done, the stop solution (100 µL) was added. The plates were then left in an insulated

7
box for 1 hour with a moist paper towel to keep the environment humid. Once the hour
incubation was finished, each well was resuspended carefully not to produce bubbles. This step
is also crucial as the dye settles and a homogeneous mixture will give more consistent results.
The absorbance of each well was then reads at 570nm. The higher the reading the more viable
the cells are.
Proliferation
EDU staining was used to study the proliferation of the MSC after treatment with Nicotine,
PGVG, and E-Cig. Cells were seeded at 20% confluency in 8 well chamber slides and left
overnight. 24 hours before staining the cells the media was then removed and treated media was
added to each perspective well. 12 hours before staining, EdU was added as well as treated
media. The cells were fixed and stained following the Click-it EdU kit and imaged for Hoechst
33342 and Alexa fluor 647. Cells signals were counted for both signals and compared for cells
and cells positive for the proliferative marker.
Calculations: Statistics performed using GraphPad prism to plot and run t-test to test for
significance.

8
Chapter Three: Results
Treatment of nicotine, PGVG, and E-Cig
Electronic cigarettes range from 0-5% making PGVG range from 100% - 95 % of the E-cig
Juice. Originally, we had decided to follow Vlasceanu et al who tested nicotine toxicity on lung
fibroblast cells. They found that viability started to decrease after 750 µM, yet we were getting
too much variability, (Figure 1) so we decided to rethink our treatment plan. To narrow down
what treatment we tested 1%, 5%, and 10% of all components (Figure 2). Although there was
some interference with the plates used a trend was still clear. We had also tried to find a positive
control unfortunately the tobacco extract was denatured. We decided to move forward with a
10% DMSO as a positive control. Unfortunately, it still did not give us a clear range to test, so
we narrowed it down more by testing few different concentrations. For nicotine and E- Cig we
tested 0.2%, 0.25% 0.5%, and 1% nicotine and for PGVG we tested 1.18%, 15%, and 30%. This
would closely mimic a 18mg/mL E-cig ratio. Within all these treatments we only saw viability in
PGVG 1.18% (Figure 3). One obstacle we had when deciding treatments was trying to stick to a
ratio of nicotine and PGVG that would be testable and yet still comparable to the vape juice sold.
Originally, we were testing a 1/59 ratio mimicking 18mg/mL. Ultimately, we decided to test Ecig starting with a stock E-cig of 5% nicotine 95% PGVG for a 1:20 ratio. The 5% nicotine E-cig
was chosen since it is to the highest concentration available commercially and the ratio of PGVG
would still be manageable to test without compromising the media and nutrients needed for the
cells to grow. Taking into consideration all the previous concentrations tested Nicotine in the
range of 0.016%, 0.05%, 0.125%, and 0.2%. The concentrations for PGVG are 0.32%, 1%, 2.5%
and 4%. E-Cig followed a combination of each designated Nicotine and PGVG tested at the 1:20
ratio. For a positive control we used 10% DMSO and untreated cells as negative control.

9
Figure 1
Figure 1. Dose dependence data- Lower concentrations. Nicotine and PGVG tested at low
concentrations following a ratio that is similar to an 18mg/mL ratio following the concentrations
tested in Vlăsceanu, A.M., et al (2018)

10
Figure 2:
Figure 2 Dose dependance data Expanded Range and hours. a) 48 hours treatment,
normalized with interference from plate b) Viability after 96 hours normalized with interference
from plate
A)
B)

11
Figure 3
Figure 3 Dose dependance data narrowing concentrations. Viability was tested using MTT
after 48hours treatment.

12
Viability
In both the GFP and RFP labeled MSC we see a trend where viability is decreasing in a dose
dependent manner for all three Nicotine, PGVG, and E-Cig (Figure 4 a-b). It was interesting to
see E-Cig treated cells with the same concentrations as nicotine and PGVG showed a higher
viability than both. Although we do not see full loss in viability for E-Cig at 0.2% in the
individual runs, after performing triplicates and we see a total loss at 0.2% and 0.5% (Figure 4c).
Overall GFP-MSC were more sensitive and even when seeded with the same initial number of
cells as RFP-MSC viability was usually lower.
Since one of the biggest selling points for E-Cigs is the different flavorings that are available.
RFP-MSC Viability was tested with several different flavorings: Vanillin (vanilla),
Cinnamaldehyde (Cinnamon), Menthol (mint) and Benzyl alcohol solution (Fruity Flavor). After
24-hour treatment with the flavoring Vanillin (1mMol) and Menthol (1mMol) viability was
significantly decreased. (Test was not replicated) (Figure 5). We did find that Cinnamaldehyde at
1% shattered and melted through the glass bottom plate. This was replicated showing both at
room temperature and at 37°C the wells with cinnamaldehyde melted through. However, when
using a plastic plate, it did not melt through (Figure 6).

13
Figure 4
Figure 4. MTT Viability after 48 hours A-B) Dose dependent decrease in viability for each
treatment in both cell lines GFP-MSC and RFP-MSC. C) Viability normalized to DMSO across
3 runs of RFP-MSC.
A)
B)
C)

14
Figure 5
Figure 5. Effects of different flavorings. RFP cells were treated with Vanillin and Menthol
were at 1mMol for 24 hours showing significant decrease in viability (p<0.0001)
Figure 6
Figure 6. Cinnamaldehyde broken plate. RFP cells were treated with cinnamaldehyde at
1mMol on glass bottom plate for 24 hours, melted through the glass after 24 hours.

15
Proliferation
For the proliferation study we decided to use two concentrations for each concentration. DMSO
10% was used as a positive control and untreated cells were used for negative control. Nicotine
0.016% and 0.125%; PGVG 0.32% and 2.5%; and for E-Cig 0.16% and 0.2% was tested. E-Cig
0.2% was tested instead of 0.125% since MTT had shown a more obvious difference at 0.2%
(Figure 7 a-h). The results show at the lower concentrations there is a slight variation in the
number of cells when compared to untreated cells. Yet when comparing to the higher
concentrations we see a significant drop in number of cells, except for PGVG cell numbers don’t
vary from one another (Figure 7I). As for the percentage of cells that can proliferate in each
group, at lower concentrations we see some slight variation. Where at the lower concentrations
Nicotine, PGVG, and E-Cig have slight decrease percent proliferative cells. At the higher
concentrations they have no signal at all for actively proliferative cells. The exception being
PGVG. In 0.32% PGVG we see less cells close to no change when comparing to untreated cells.
Although we still see PGVG have some proliferation and cells after 48 hours, we still see it start
to decrease in what appears to be a dose dependent manner. (Figure 7J). The remaining cells can
be determined by two factors: proliferation and apoptosis.

16
Figure 7

Figure 7. EdU assay to determine cell proliferation . (A-H) Hoechst 33342 staining DNA
content expressed in Blue and Alexa Fluor® 647 which is expressed when “clicked” with the
Edu that is incorporated into DNA during active DNA synthesis in cells expressed Green. (I)
percent of viable cells present in each well when compared to untreated cells. (J) % of cells that
in each well still actively proliferating.
I) J)
A)Untreated B)DMSO 10%
C) PGVG 0.32% D) PGVG 2.5%
E) Nicotine 0.016% F) Nicotine 0.125%
G) E-Cig 0.016% H) E-Cig0.2%

17
Chapter Four: Discussion
Viability
The results suggest a dose-dependent decrease in viability for all components that make up E-cigs
and the combination. However, the decrease in viability is not as extensive in E-Cigs and PGVG
when compared to nicotine alone. Overall PGVG showed less toxicity than both E-Cig and
nicotine. Overall, the trend stayed the same and we were able to find a range where all the three
components can be tested to show dose dependance and not just total loss. Finding this range will
help with future studies. One flaw in this testing was increasing ratio of PGVG when nicotine
concentrations decrease was not tested. The combination of E-Cig with flavoring was not tested to
compare if there is a difference between toxicity of non-flavored versus E-Cig flavored E-Cigs.
Proliferation
The results of the proliferation study also suggest a dose dependent effect on proliferation of the
MSCs. However, PGVG does show less toxicity to the MSC allowing for a higher percentage of
the cells to show proliferative properties. However, when combined into the E-Cig juice we see
that the negative effect of nicotine outweighs the lesser effect of PGVG.
General
The effects of smoking nicotine and tobacco products is already well known to be harmful. When
it comes to the use of E-cigarettes there is still a lot that is not known, especially long-term effects.
It becomes more concerning when many people see the use of E-Cigs as a better alternative for
long term use not understanding how toxic E-cigs are. Having E-Cigs not only damage lung tissue
but also having it damage the lung mesenchymal stem cells is very concerning as they aid in the
repair of other damaged cells. There is still a lot that needs to be studied when it comes to E-cig
toxicity on MSC especially when it comes to higher concentrations. The concentrations tested here

18
found significant decrease in viability and proliferation at concentrations of nicotine that are
considered low in most electronic cigarettes. The highest tested was 0.2% which is 2mg/mL the
highest sold is 5% or 50mg/mL. Overall nicotine concentration seems to be more of toxic
compared to PGVG.

19
Chapter 5: Future Study
Future test will include testing if E-Cigs affect differentiation of these lung mesenchymal stem
cells. As the lung mesenchymal stem cells can differentiate into cells such as adipocytes,
osteoblast, and chondrocytes) in vitro [3] (Dominici et al., 2006). This will be done by
differentiating the MCS through Adipogenesis and osteogeneses in tangent with treatment of ECigs at differing concentrations. Studying the effects of E-cig smoke would also be a future study
as we did not test the effects after the PGVG, and nicotine was vaporized. Looking into the
chemical components after heating will help us better understand the chemicals effects when
smoked as when chemicals are heated together, we at times see different reactions. That will further
help understand not only the effects of E-cigs chemical components but also the effect of the cotton
and coil the E-cigs use to vaporize the E-cigs. Due to a lack of a standard material for coils and
wicks, testing different vaporizing mechanisms will help us understand if there is any difference
in the toxicity of E-cigs when smoked using devices made with differing types of materials.

20
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https://doi.org/10.1186/1741-7007-11-111

Abstract (if available)

Abstract For years now electronic cigarettes have been promoted as a better alternative to smoking cigarettes, but the effects of electronic cigarettes have not been fully explored. Smoking can lead to diseases such as Chronic obstructive pulmonary disease (COPD). When tissues in the body are damaged, such as the lungs, mesenchymal stem cells (MSC) help repair and regenerate cells. Understanding the effects of electronic cigarettes on the lung mesenchymal stem cells will help us understand the relationship between smoking electronic cigarettes and diseases such as emphysema and chronic bronchitis. The MSCs help keep the lungs in homeostasis and aid in organogenesis. If the viability is decreasing due to the exposure to E-Cigs, then the MSCs will not be able to continue to aid in homeostasis. Using Lung mesenchymal stem cells from triple transgenic mice we were able to test the viability and proliferation of Tbx4 lineage labeled fetal lung-derived MSCs and Tbx4 negative MSCs.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Siu, Krysta Marie (author)

Core Title Effects of electronic cigarettes on viability and proliferation of lung mesenchymal stem cells

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Degree Conferral Date 2023-12

Publication Date 11/03/2023

Defense Date 11/02/2023

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag electronic cigarettes,mesenchymal stem cells

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Lien, Ching-Ling (Ellen) (committee chair), Frey, Mark (committee member), Rice, Judd (committee member)

Creator Email kmsiu@usc.edu,krystasiu11@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC113763528

Unique identifier UC113763528

Identifier etd-SiuKrystaM-12452.pdf (filename)

Legacy Identifier etd-SiuKrystaM-12452

Document Type Thesis

Format theses (aat)

Rights Siu, Krysta Marie

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Source 20231107-usctheses-batch-1105 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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